A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis
© Stump et al.; licensee BioMed Central Ltd. 2011
Received: 31 August 2010
Accepted: 21 April 2011
Published: 21 April 2011
Janus kinase 2 (JAK2) is involved in the downstream activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 and is responsible for transducing signals for several proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), including interleukin (IL)-6, interferon γ (IFNγ) and IL-12. In this paper, we describe the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 evaluated in two mouse models of RA.
Collagen antibody-induced arthritis (CAIA) and collagen type II (CII)-induced arthritis (CIA) were established before the oral administration of a small-molecule JAK2 inhibitor, CEP-33779, twice daily at 10 mg/kg, 30 mg/kg, 55 mg/kg or 100 mg/kg over a period of 4 to 8 weeks.
Pharmacodynamic inhibition of JAK2 reduced mean paw edema and clinical scores in both CIA and CAIA models of arthritis. Reduction in paw cytokines (IL-12, IFNγ and tumor necrosis factor α) and serum cytokines (IL-12 and IL-2) correlated with reduced spleen CII-specific T helper 1 cell frequencies as measured by ex vivo IFNγ enzyme-linked immunosorbent spot assay. Both models demonstrated histological evidence of disease amelioration upon treatment (for example, reduced matrix erosion, subchondral osteolysis, pannus formation and synovial inflammation) and reduced paw phosphorylated STAT3 levels. No changes in body weight or serum anti-CII autoantibody titers were observed in either RA model.
This study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA. Using a selective inhibitor of JAK2 rather than pan-JAK inhibitors avoids the potential complication of immunosuppression while targeting critical signaling pathways involved in autoimmune disease progression.
Inflammation acting as a driver of pathology has previously been thought to be primarily restricted to infectious diseases and autoimmune disorders; however, it has become more apparent that inflammation plays a larger role in multiple different disease indications, such as obesity, coronary artery disease and cancer [1, 2]. Targeting mediators of inflammation has received enormous attention in the past decade with the advent of biologics, most notably antibodies that capture or neutralize disease-driving cytokines . However, biologics have several disadvantages compared with orally active small molecules, such as route of administration, cost of production and the possibility of anaphylactic reactions . Unlike the direct targeting of cell surface receptors or cytokines, another approach is to target cytokine pathways by inhibiting key kinases involved in transducing cytokine receptor signals . In the present article, we describe the preclinical evaluation of a potent, orally active, small-molecule inhibitor of Janus kinase 2 (JAK2), CEP-33779, for the treatment of rheumatoid arthritis (RA).
RA is a chronic systemic inflammatory disorder that primarily affects the synovial joints. RA can also produce diffuse inflammation in the lungs and pleura and also forms subcutaneous nodular lesions. About 1% to 2% of the world's population is affected by RA, and there is a gender bias associated with disease onset. Women are three times more likely than men to develop RA between the ages of 35 and 50 years. Treatments for RA have included mainly disease-modifying antirheumatic drugs and have been more prolific than those for the more difficult to treat rheumatic diseases, such as lupus. Approved treatments for RA include nonsteroidal anti-inflammatory drugs, antimetabolites such as methotrexate and leflunomide, various corticosteroids and glucocorticoids, sulfasalazine, and various biologics, including abatacept, adalimumab, etanercept, infliximab, golimumab, and rituximab . Other molecules implicated in the pathogenesis of RA include B lymphocyte stimulator/B cell activating factor, a proliferation-inducing ligand, p38/mitogen-activated protein kinase, and the B-cell receptor protein spleen tyrosine kinase Syk [6, 7]. The recent US Food and Drug Administration approval of tocilizumab (anti-interleukin (IL)-6R) further demonstrates the power of targeting cytokines and associated receptors to treat chronic inflammatory diseases .
Using small molecules to target cytokine pathways is an attractive method to treat disease simply because of the oral bioavailability of small molecules and the reduced cost of production, thus reducing the cost carried over to the patient and healthcare system as a whole. The safety profile of antibody biologics, especially anti-tumor necrosis factor α (anti-TNFα) antibodies, has come under greater scrutiny over the past several years as more and more patients and physicians have reported problems associated with long-term use [9–11]. It is well-documented that JAK kinases play a pivotal role in cytokine receptor signaling to phosphorylate and activate signal transducer and activator of transcription (STAT) proteins. Several of these JAK-controlled cytokine receptor pathways are intimately involved in the initiation and progression of RA disease pathogenesis . Cytokines involved in these diseases (for example, interleukin (IL)-2, IL-6, IL-12, interferon γ (IFNγ) and granulocyte macrophage colony-stimulating factor (GM-CSF)) are also essential to a properly functioning immune response to infectious agents. JAK1, JAK2 and tyrosine kinase 2 (TYK2) kinases are ubiquitously expressed, whereas JAK3 is limited to the lymphoid lineage as JAK3 associates with the common γ-chain.
The therapeutic benefit of JAK kinase inhibition has already been established in RA with the use of CP-690,550, a pan-JAK inhibitor originally intended for organ transplantation immunosuppression, as it is a potent inhibitor of JAK3 but has also been shown to have activity against JAK1 and JAK2 . More recently, the selective JAK1/JAK2 inhibitor INCB028050 has been shown to have efficacy in various rodent models of RA, further demonstrating the central role that JAK kinase plays in this disease . The mechanistic rationale of targeting cytokine pathways central to the pathogenesis of RA, but without the immunosuppressive side effects of several of the TNFα blockade biologics, has been demonstrated in the successful use of tocilizumab to treat RA . The present report shows that a novel, potent, orally active, selective JAK2 inhibitor, CEP-33779, can ameliorate disease in two mouse models of RA. JAK2 inhibition decreased several inflammatory cytokines, including IL-6, IL-12, IL-1β and IFNγ, both systemically and locally at the site of disease and treated symptoms of chronic, pathological joint destruction in a well-tolerated manner.
Materials and methods
Animals, antibodies, cell culture reagents, compounds, cell lines
Mice used for collagen-induced arthritis (CIA) and collagen-antibody induced arthritis (CAIA) studies were Harlan female DBA/1 mice (Harlan, Somerville, NJ, USA) Female Balb/c mice from Jackson Laboratories (Bar Harbor, ME, USA) were used for all air pouch model (APM) studies. Mice were age-matched 6 to 8 weeks from the start of the experiments. All animals were maintained on a 24-hour light-dark cycle, with food and water available ad libitum. All experimental animal procedures were approved by, and in accordance with the regulations of, the Institutional Animal Care and Use Committee of Cephalon, Inc.
CEP-33779 was prepared in a manner analogous to the five-step method described elsewhere . Compound CEP-33779 was administered orally in suspension form, dissolved in dimethyl sulfoxide (DMSO) (1% final concentration) (Sigma, St. Louis, MO, USA), and further reconstituted in PEG400 (Emerald BioSystems, Bainbridge Island, WA, USA) to the desired concentration for oral administration. The compound was administered orally through gavage tubes for all animals treated with CEP-33779.
All spleen samples were analyzed using an Accuri C6 Flow Cytometer (Accuri Cytometers, Ann Arbor, MI, USA). Antibodies used for Western blot analysis were anti-phosphorylated STAT5 (Cell Signaling Technology, Danvers, MA, USA) and anti-total STAT5 (Cell Signaling Technology). Complete media (R10) was used for all experiments involving the ex vivo culture of splenocytes for all enzyme-linked immunosorbent spot assay (ELISPOT) experiments. Complete media consisted of RPMI 1640 (Cellgro, Manassas, VA, USA) plus 1% Pen-Strep (Cellgro), 1% L-Gln (Cellgro), 1% nonessential amino acids (Cellgro), β-mercaptoethanol (Sigma), and 10% fetal bovine serum (Cellgro). The small-molecule JAK2 inhibitor CEP-33779 was synthesized according to procedures detailed elsewhere . The cell line HEL92 (American Type Culture Collection, Manassas, VA, USA) was used for early pharmacodynamic (PD) testing of phosphorylated STAT5 (pSTAT5) inhibition using CEP-33779.
Western blot analysis
Samples were heated at 70°C for 10 minutes and loaded onto a NuPAGE 10% Bis-Tris gel (1.5 mm, 15 wells; Invitrogen, Carlsbad, CA, USA). The molecular weight marker used was a Bio-Rad kaleidoscope prestained standard (Bio-Rad, Hercules, CA, USA). Run conditions were 150 V for 1 hour at room temperature (RT). Using a semidry transfer apparatus (Bio-Rad), proteins were transferred onto a nitrocellulose membrane. Primary antibody diluted at 1:1,000 in 5% milk for pSTAT3 pY705 (Cell Signaling Technology) and total STAT3 (Cell Signaling Technology) diluted to 1:1,000 in 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) were incubated overnight at 4°C with 5% milk in TBS plus 0.05% Tween 20 (TBS-T). Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked secondary antibody was used at 1:2,000 dilution (Cell Signaling Technology). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA). Densitometry was performed using Gel-Pro Analyzer 3.1 software (Media Cybernetics, Inc., Bethesda, MD, USA).
The kinase activity of baculovirus-expressed human JAK kinases (JAK1, JAK2, JAK3 or TYK2) was measured using the time-resolved fluorescence detection system as described elsewhere . Each 96-well Costar high binding plate (Corning, Corning, NY, USA) was coated with 100 μl/well of 10 μg/ml neutravidin (Pierce Biotechnology) in TBS at 37°C for 2 hours, followed by 100 μl/well of 1 μg/ml 15-mer peptide substrate (biotinyl-amino-hexanoyl-EQEDEPEGDYFEWLE-amide; Infinity Biotech Research and Resource, Aston, PA, USA) at 37°C for 1 hour. The kinase assay mixture (total volume 100 μl/well), consisting of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), adenosine triphosphate (ATP) (Km level for each kinase), 1 mM MnCl2, 0.1% BSA, and test compound (diluted in DMSO; 2.5% DMSO final concentration in assay), was added to the assay plate. The concentrations of ATP used were as follows: 0.2 μM ATP for JAK1, JAK2, and TYK2 and 0.1 μM ATP for JAK3. Enzyme was added, and the reaction was allowed to proceed for 20 minutes at RT. Detection of the phosphorylated product was performed by adding 100 μl/well of Eu-N1-labeled PY100 antibody (PerkinElmer Life Sciences, Boston, MA, USA) diluted 1:5,000 or 1:10,000 in 0.25% BSA in TBS-T. Samples were incubated at RT for 1 hour, followed by the addition of 100 μl of enhancement solution (PerkinElmer Life Sciences). Plates were agitated for 10 minutes, and the fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 or 2104 multilabel plate reader. Half maximal inhibitory concentration (IC50) values were determined using the four-parameter logistic model in XLFit 4 (IDBS, Ltd., Guildford, UK).
Acclimated mice were randomized, prebled and measured for baseline inflammation before any studies were initiated. On day 0, DBA/1 female mice were injected intravenously (i.v.) with 100 μl of a saline solution containing 1.5 mg of a 10 mg/ml cocktail of arthritogenic monoclonal antibodies directed against different epitopes of CII (Chondrex, Redmond, WA, USA) suspended in saline solution. On day 3, mice were injected intraperitoneally (i.p.) with 100 μl of 50 μg of Escherichia coli 0111:B4 lipopolysaccharide (LPS) (0.5 mg/ml stock) (Chondrex) and fed gel food during recovery. Two days after LPS treatment monitoring of arthritic paws began. Peak arthritis usually occurred 2 to 3 days following LPS treatment. To ensure that enough mice were available for each study, a surplus of animals were induced, and only those mice that met the criteria were entered into the study. Mice that exhibited a clinical score greater than 1 in each limb were considered arthritic and were entered into the study. If mice did not meet the study criteria, they we excluded and killed. Mice were then randomized and ear-tagged for identification. Study group sizes were determined after study entry, but consisted of at least 10 mice per group. Arthritis clinical score tables can be found elsewhere . Briefly, a score of 0 denotes no evidence of erythema and swelling, 1 denotes erythema and mild swelling confined to the tarsus, 2 denotes erythema and mild swelling extending from the ankle to the midfoot, 3 denotes erythema and moderate swelling extending from the ankle to the metatarsal joints, and 4 denotes erythema and severe swelling encompassing the ankle, foot and digits. The black arrows in the respective figures indicate treatment start. CEP-33779 compound was administered orally (p.o.) by gavage instrument suspended in vehicle twice daily (b.i.d.). Vehicle consisted of PEG400 plus 1% DMSO administered p.o., b.i.d., and dexamethasone (Dex) (Hanna's Pharmaceutical Supply, Wilmington, DE, USA) at 1.5 mg/kg was administered i.p. three times weekly in saline solution and continued from the peak of inflammation until the end of the experiment. Mice were cheek-bled for serum sample collection for cytokines and antibodies throughout the experiment. All samples were kept at -80°C until ready to assay. At about 4 weeks, mice were harvested for spleen, serum and arthritic paws for STAT and cytokine quantitation and histological analysis. For earlier validation, studies were ended around 3 to 4 weeks after induction, and the paws were removed at various time points to measure cytokine levels over time.
Acclimated mice were randomized, prebled and measured for baseline inflammation before any studies were initiated. On day 0, DBA/1 female mice were given a primary immunization with equal volumes of bovine CII, 2 mg/ml stock in 0.05 M acetic acid (Chrondrex) suspended in 5 mg/ml Complete Freund's Adjuvant (CFA) (Chondrex) emulsion, yielding an injection of 100 μg of CII at 100 μl into the base of the tail intradermally (i.d.). At day 21 post-primary immunization, mice were rechallenged with CII emulsified in Incomplete Freund's Adjuvant (IFA) (Thermo Scientific, Rockford, IL, USA) subcutaneously (s.c.) on the flank of the mouse. On day 28, each mouse received an i.p. injection of E. coli 0111:B4 LPS (0.5 mg/ml stock) (Chondrex) at 10 μg in 100 μl of saline solution (25 to 50 μg of LPS reduced to 10 μg per mouse to reduce mortality associated with endotoxic shock). Around day 35 the mice begin to show signs of paw inflammation and disease. Before the start of treatment, mice were randomized, grouped, scored, ear-tagged and prebled to determine baseline disease. To ensure that enough mice were included in each study, a surplus of animals were induced, and only those mice that met the study criteria were entered into the study. Mice that exhibited a clinical score greater than 1 in each limb were considered arthritic and were entered into the study. If mice did not meet the study criteria, they were excluded and killed. Mice were then randomized and ear-tagged for identification. Study group sizes were determined after study entry, but consisted of at least 10 mice per group. Arthritis clinical scores were the same as those used to score the mice in the CAIA model (see CAIA model). The black arrows in the respective figures indicate treatment start. CEP-33779 compound was administered orally by gavage suspended in vehicle b.i.d. Vehicle consisted of PEG400 plus 1% DMSO p.o., b.i.d., and 1.5 mg/kg Dex (Hanna's Pharmaceutical Supply) was administered i.p. three times weekly in saline solution and continued from the peak of inflammation until the end of the experiment. Mice were cheek-bled for serum sample collection for cytokines and antibodies throughout the experiment. All samples were kept at -80°C until ready to assay. At about day 60, mice were harvested for spleen, plasma and arthritic paws for STAT and cytokine quantitation and histological analysis.
APM for carrageenan-induced inflammation
Details on the establishment and design of this rapid inflammation screening model can be found elsewhere [19, 20]. Briefly, Balb/c mice were injected s.c. with 4 ml of sterile air on day -5, baseline measurements were recorded, and mice were randomized into groups. Mice entered the study upon air pouch maintenance by day -3, when 2 ml of sterile air was injected for air pouch maintenance. On day 0, mice were given a 1% carrageenan (Sigma) injection suspended in saline solution directly into the air pouch. This induced a rapid inflammatory influx of primarily neutrophils and macrophages (data not shown). Compound was administered in saline solution at milligrams per kilogram concentrations in 100 μl total volume directly into the air pouch simultaneously with carrageenan injection (1% carrageenan in saline solution). The response was followed by a spike in IL-6, TNFα, and IL-1β pouch cytokines (data not shown) and was completed by day 8 (data not shown). Twenty-four hours after carrageenan and compound administration, 100 μl of air pouch fluid was removed along with cellular exudates. Cellular exudates were centrifuged, the supernatants were processed for Luminex cytokine analysis (Invitrogen), and the cell pellets were counted for total live and dead cells. Total cell counts were performed using BD TruCount beads (BD Biosciences, San Jose, CA, USA), and cellular analysis was performed using 7-aminoactinomycin D (Sigma) as a viability dye to exclude dead cells. Hemoglobin content was determined using the Drabkin's assay (Sigma).
CAIA and CIA arthritis models
At the end of each experiment, front and hind paws (including carpus and tarsus) were removed from the body of each animal for histological analysis. Skin from the ends of the digits was removed, and the metatarsal region skin was perforated using surgical scissors to allow full decalcification. Samples were treated using a decalcification procedure with formic acid as described elsewhere [21, 22]. After 7 to 10 days of decalcification, samples were washed for 2 hours in distilled water and stored in 70% ethanol at 4°C until they were ready to be processed. Samples were paraffin-embedded, sectioned, and stained with hematoxylin and eosin as well as Safranin O for matrix degradation (images not shown, used by pathologist for scoring). All histological work was performed at the Wistar Institute (Philadelphia, PA, USA). Images were collected using an Olympus BX50 microscope equipped with an Olympus DP70 camera and Olympus LabSens software (Olympus, Center Valley, PA, USA). A total of five mice from each group were analyzed via histopathology, and representative × 10 original magnification images are shown for each group tested.
Measurement of serum anti-collagen type I and anti-CII autoantibodies
Serum was collected every 1 to 2 weeks and stored at -80°C until use. Thawed samples were analyzed by using an in-house-generated CII and collagen type I (CI) enzyme-linked immunosorbent assay (ELISA) method. Briefly, each well of a 96-well plate was coated with 50 μl of 5 μg/ml CI (Chondrex) or CII (Chondrex) in borate-buffered saline (BBS) (0.025 M Na2B4O7-10H2O, 0.01 M H3BO3, and 0.075 M NaCl; pH 8.4) buffer overnight at 4°C. Plates were washed with BBS plus 0.1% Tween 20 three times before proceeding. Standard curves were generated using purified mouse anti-CI or anti-CII antibody, and serum was added at 50 μl per well and then incubated at RT for 1 hour followed by being washed with BBS plus Tween 20. After being washed, 50 μl of 1 μg/ml rabbit anti-mouse HRP-fragment antigen-binding antibody was added as a detection antibody (Rockland Biologicals, Gilbertsville, PA, USA) and incubated at RT for 45 minutes. Plates were washed four times with BBS plus Tween 20, then 100 μl of tetramethylbenzidine substrate was added and the reaction was stopped using 100 μl of 1 M H2SO4. The plates were read at 450 nm with a reference wavelength of 570 nm.
T-cell ELISPOT assays
For CIA experiments, Millipore nitrocellulose IP filter plates (Millipore, Billerica, MA, USA) were coated with either anti-mouse IFNγ (AN18; MabTech, Mariemont, OH, USA) or anti-mouse IL-4 (11B11; eBioscience, San Diego, CA, USA) at 7 μg/ml or 2 μg/ml, respectively, in sterile phosphate-buffered saline (PBS) at 60 μl/well and incubated for 3 hours at RT or overnight at 4°C. Coated wells were washed with PBS plus 0.1% Tween 20 and patted semidry before blocking using complete media for at least 1 hour before use. Purified mouse CII (Chrondrex), CI (Chrondrex), or third-party control chicken ovalbumin (OVA) (Sigma) was used to challenge 1 million processed splenocytes per well at 10 μg/ml total antigen concentration. Media alone was used as a background control, OVA was used as a third-party control, and phorbol 12-myristate 13-acetate plus ionomycin was used as a positive control for IFNγ and IL-4 release by T helper 1 (Th1) and Th2 cells, respectively.
B-cell ELISPOT assays
B-cell ELISPOT components were obtained from MabTech, and nitrocellulose IP filter plates were obtained from Millipore. Briefly, for CIA experiments, B-cell ELISPOT wells were activated using ethanol, then washed and coated with 10 μg/ml purified bovine CII or purified bovine CI (Chrondrex) in sterile PBS. OVA was used as a third-party control, and media alone was used for background. Processed splenocytes were added to each well and were not stimulated with LPS to avoid true ex vivo frequencies of antibody-secreting cell types (ASCs). Anti-mouse total IgG was used as a positive control for total IgG-producing ASCs and was used to normalize results. Cell frequencies for the detection of each antigen were previously identified during optimization and validation phases for each model tested (data not shown). A total of 500,000 cells were added to each well for the CI- and CII-coated wells for CIA experiments. B-cell ELISPOT spots were incubated overnight at 37°C and 5% CO2. To develop each assay, a secondary antibody (MabTech) was added to each well, incubated, and washed; alkaline phosphatase streptavidin was used as a conjugate (Jackson Immunoresearch, West Grove, PA, USA); and 5-bromo-4-chloro-3'-indolylphospate ρ-toluidine salt (BCIP) and nitro-blue tetrazolium chloride (NBT) (Rockland Biologicals) was used as a substrate and developed until spots were visible. All ELISPOT analyses were performed using a CTL ImmunoSpot Analyzer and CTL BioSpot software (Cellular Technology Ltd., Shaker Heights, OH, USA).
Measurement of serum cytokines via multiplex Luminex bead assays
Frozen plasma at -80°C was thawed on ice, vortexed, and then centrifuged for 10 minutes to remove debris and aggregates. A total of 25 to 50 μl of serum were used for Luminex assays (Invitrogen) following the manufacturer's instruction. Ten different mouse cytokines were measured using the mouse cytokine 10-plex bead kit (Invitrogen). Briefly, filter plates (Millipore) were prewetted with 200 μl of wash solution (kit component), and 25 μl of beads were added per well. Serum samples were diluted, and a total volume of 50 μl/well was added (that is, 25 μl of sample serum plus 25 μl of assay diluent). Plates with beads were incubated for 2 hours at RT on an orbital shaker in the dark. At the end of the incubation, the plates were washed twice in buffer and 100 μl of secondary biotinylated antibody were added at a 1:10 dilution in biotin diluent provided with the kit. Plates were incubated at RT for 1 hour in the dark, then washed twice in buffer. Streptavidin in assay diluent was added at 100 μl/well, then incubated for 30 minutes at RT in the dark. Plates were washed three times with 100 μl of wash solution and agitated for 2 to 3 minutes at RT in the dark. Plates were run immediately on a Luminex xMAP 200 unit with data acquisition and analysis software (Invitrogen). All bead washing was performed using a vacuum manifold unit (Pall, Port Washington, NY, USA).
Measurement of paw cytokines and pSTAT levels via multiplex Luminex bead assays
The method described in this subsection has been published elsewhere . Briefly, the front (including carpus) and back (including tarsus) right paws were collected, frozen in dry ice-cooled isopentane, and stored at -80°C until use. The paws were cut to 2 mm × 2 mm segments and kept on dry ice in round-bottomed polypropylene tubes. Seven hundred microliters of tissue extraction buffer containing protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and Halt phosphatase inhibitor cocktail (Thermo Scientific) or Roche phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA) in tissue extraction reagent I (Invitrogen) were added to each sample. Samples were homogenized frozen using a PT 10/-35 Polytron Homogenizer (Brinkmann, Dallas, TX, USA). After homogenization, samples were centrifuged at 4°C and 2,000 × g for 10 minutes, and the supernatants were recentrifuged at 4°C at maximum speed, 14,000 × g, for 15 minutes. Supernatants were carefully removed to avoid picking up the top layer of lipids/adipose debris. The protein concentration was measured using a bicinchoninic acid protein assay (Pierce Biotechnology) and adjusted to 3 mg/ml. A minimum of 25 μl of this extraction was used in the above-described protocol using the mouse cytokine 10-plex bead kit (Invitrogen) or STAT1, 3, 5a/b Phospho 3-Plex Panel (Invitrogen).
Compound quantification (Pharmacokinetics)
Plasma and protein extracted from paw and spleen samples were submitted for quantitative analysis to determine the respective compound concentrations. Blood samples were collected into heparinized tubes and placed on wet ice until being centrifuged (16,000 × g for 5 minutes) to separate the plasma. Supernatant was collected and stored at -20°C pending analysis. At the time of analysis, two volumes of cold acetonitrile containing an internal standard (alprenolol) were added to each sample, which were then vortexed and centrifuged. The supernatants were removed and placed into an autosampler vial, and the amount of compound present in the samples was analyzed by liquid chromatography/mass spectrometry. The concentration of compound in the samples was quantified against a mouse plasma standard curve made via serial dilution in a concentration range from 5 to 20,000 ng/ml. Samples containing concentrations more than 10% above the top of the standard curve were diluted 1:10 with acetonitrile. The limits of detection for plasma, paw, and spleen were <10 ng/ml.
All ELISA or Luminex assays were analyzed using linear regression curves to determine the concentration of analyte following data acquisition. Mann-Whitney nonparametric one- or two-way analysis of variance (ANOVA) was used as for statistical testing as noted in the figure legends, depending on the experiment and the tested hypothesis. Bonferroni posttests were used to compare replicates in ANOVA tests. A P value < 0.05 was considered significant. For all studies, a minimum of 10 mice were included in each experimental group. The statistical software used was GraphPad Prism version 5.01 (GraphPad, La Jolla, CA, USA), and calculations were performed using Microsoft Excel software (Microsoft Corp., Redmond, WA, USA).
In vitro and in vivo characterization of the JAK2 inhibitory activity of CEP-33779
JAK2 inhibition suppresses CAIA and inhibits local cytokine responses
To investigate the effect of CEP-33779 on levels of pSTAT and cytokines in the paws of treated mice, a method was developed whereby synovial fluid and cellular joint extracts could be obtained from the paws and ankles of diseased animals for analysis using multiplex bead arrays . On the basis of the activity of CEP-33779 as a JAK2 inhibitor, pSTAT5, pSTAT3, and possibly pSTAT1 were hypothesized to be reduced in the paws of treated mice as previously suggested on the basis of in vitro and in vivo experiments (Figures 1A and 1B and data not shown). Using multiplex bead kits, it was determined that measurement of pSTAT was similar and more quantitative than using Western blot analysis alone. Consequently, multiplex analysis was used as the primary method of measuring pSTAT for all further studies reported here .
CAIA model joint inflammation histopathology scoresa
1.55 ± 0.07
3.12 ± 0.71
2.50 ± 1.00
1.65 ± 0.47
2.00 ± 0.81
1.12 ± 0.85
1.75 ± 0.95
CEP-33779 10 mg/kg
1.39 ± 0.38
3.40 ± 2.07
2.30 ± 1.30
1.70 ± 1.09
2.40 ± 1.34
1.30 ± 0.97
1.60 ± 1.14
CEP-33779 30 mg/kg
1.12 ± 0.88
0.75 ± 0.95
0.62 ± 0.47*
0.87 ± 0.62
0.25 ± 0.50*
0.00 ± 0.00
0.12 ± 0.25*
CEP-33779 55 mg/kg
2.05 ± 0.07
0.70 ± 0.44*
0.60 ± 0.41**
0.30 ± 0.72*
0.30 ± 0.44*
0.00 ± 0.00*
0.20 ± 0.27*
Dex 1.5 mg/kg
0.98 ± 0.44
1.00 ± 0.79
0.50 ± 0.35**
0.70 ± 0.44
0.40 ± 0.54*
0.10 ± 0.22
0.30 ± 0.44*
Compared to vehicle
*P < 0.05
**P < 0.01
***P < 0.001
Inhibiting JAK2 slows the progression of chronic CIA and reduces the generation of disease-promoting Th1 cells
CIA model joint inflammation histopathology scoresa
3.00 ± 0.00
3.67 ± 1.52
2.50 ± 0.50
2.66 ± 0.57
2.66 ± 0.57
1.66 ± 0.57
2.83 ± 0.28
Compound A 30 mg/kg
2.50 ± 0.57
4.62 ± 0.75
2.75 ± 0.50
2.50 ± 0.40
2.75 ± 0.50
2.37 ± 0.94
2.12 ± 0.25
Compound A 55 mg/kg
1.83 ± 0.76
2.83 ± 1.60
1.33 ± 1.04
1.83 ± 1.15
1.00 ± 0.86
0.83 ± 0.76
0.66 ± 0.57*
Compound A 100 mg/kg
1.83 ± 0.28
2.33 ± 1.52
1.50 ± 0.50
1.66 ± 0.57
1.50 ± 0.86
0.66 ± 1.15
1.33 ± 0.57**
Dex 1.5 mg/kg
2.25 ± 0.95
3.12 ± 2.09
1.37 ± 0.94
1.50 ± 1.08
1.25 ± 0.86
0.75 ± 0.64
1.00 ± 0.81**
Compared to vehicle
*P < 0.05
**P < 0.01
***P < 0.001
Inhibition of cellular immunity with JAK2 inhibition using an air pouch inflammation model
Here we report the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 that we evaluated in two mouse models of RA. PD inhibition of JAK2 reduced mean paw edema and clinical scores in both the CIA and CAIA models of arthritis. Reduction in paw and serum cytokines correlated with reduced spleen CII-specific Th1 cell frequencies as measured by ex vivo IFNγ ELISPOT assays. Both models demonstrated histological evidence of disease amelioration upon treatment and reduced paw pSTAT3 levels. This study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA.
The CIA model is dependent on both T- and B-cell responses and, as in human RA, depends on the action of IL-6, demonstrating that CEP-33779 could modulate key immune cytokines involved in human disease [15, 37, 38]. Unlike the CIA model, the CAIA model has been shown not to be entirely dependent on IL-6, as arthritis can still be induced in mice deficient in this cytokine . However, surprisingly, robust activity of CEP-33779 was observed in the CAIA model, sometimes exceeding that of the standard-of-care agent Dex. The use of tocilizumab demonstrates how targeting cytokines can directly affect disease progression without overt immunosuppression . Other small molecules such as INCB028050 have also been reported to show a similar phenotype, that is, inhibition of IL-6 and disease without immunosuppression . It is possible that CEP-33779 works similarly to INCB028050, as both can inhibit immune cellular flux and cytokine release in acute inflammation mouse models (that is, delayed hypersensitivity for INCB028050 and the APM model for CEP-33779) (Figure 5).
JAK2 is responsible for mediating signaling through several receptors, including those highly implicated in playing an essential role in autoimmunity and inflammation, such as the gp130 receptor family members IL-6R and IL-12Rβ and IFNγR2 . Blockade of JAK2 associated with these major cytokine receptors may affect the subsequent expression or production of downstream cytokines during arthritis and may be responsible for some of the results shown here. For example, reduction of cytokines IL-12, IL-2, and TNFα may switch the local milieu from a strong Th1/Th17 phenotype to a more immunoregulatory Th2/regulatory T cell phenotype and lead to disease symptom reduction .
Interactions of JAK2 with various immune-related signaling molecules will determine how inhibition of this kinase may affect other immune cell types and thus the treatment of other autoinflammatory diseases. Downstream JAK2 can interact with STAT5a/b, STAT3, STAT4, and STAT1 . However, only STAT5a/b are directly activated by JAK2; STAT4 can be activated via TYK2, STAT1 can be activated via JAK1, and STAT3 can be activated via the EGFR pathway and JAK1 and Src kinases [28, 29]. In addition, several kinases, including Tec, Vav1, Fyn, Yes1, and protein tyrosine kinase 2, also directly interact with JAK2 [43–47]. These kinases are all involved at some level in the regulation and activation of immune subsets, especially those of T and B lymphocytes. Inhibiting JAK2 may have implications other than just direct inhibition of cytokine signaling. Downstream inhibition of T- and B-cell activation may also result from JAK2 inhibition, expanding the possibilities of using a potent inhibitor of this kinase in alternate indications.
Suppressors of the JAK-STAT signaling pathway, suppressors of cytokine signaling (SOCS), also interact directly with JAK2 (for example, SOCS3) [42, 48]. SOCS proteins act as a negative feedback loop for the JAK-STAT pathway . Interestingly, the expression of SOCS is altered in RA patients; levels of SOCS1 and SOCS3 were shown to be increased in peripheral blood mononuclear cells (PBMCs) of RA patients . SOCS1 was upregulated in peripheral blood T cells associated with SOCS3 expression in PBMCs in patients with RA . SOCS1 was also showed to be upregulated in the synovial membranes of patients with RA compared to patients with osteoarthritis. This suggests that deregulation of JAK2 signaling via effects on SOCS can contribute to RA development and may predict a patient's susceptibility to disease development.
This report demonstrates the use of a highly selective and potent JAK2 inhibitor, CEP-33779, for the treatment of RA using two different mouse models of RA. Inhibition of disease-promoting cytokine pathways by CEP-33779 nearly rivaled that of standard-of-care treatments, strongly suggesting the use of this inhibitor in other IL-6-dependent diseases, such as Crohn's disease and ulcerative colitis. CEP-33779 may also have utility in other diseases dependent on the same pathways such as systemic lupus erythematosus, inflammatory bowel disease, psoriasis, and various cancers, including colon, prostate, and pancreatic cancer. These results warrant further preclinical evaluation of selective JAK2 inhibitors such as CEP-33779.
type II collagen
collagen antibody-induced arthritis
We thank the following individuals who helped make this paper possible: Russell Delgiacco at the Wistar Institute for histology work; Julie Engiles at the University of Pennsylvania New Bolton Center for pathology scoring and image results; Lisa Aimone and Deborah Galinis for PK evaluations; and Sheryl Meyer, Chrysanthe Spais, Beverly Holskin, Kristen Murray and John Durkin for the cloning and expression of JAK2 and related proteins for compound evaluation.
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